Polypeptide growth factors mediate their physiological responses by binding cell surface receptors with tyrosine kinase enzymatic activity (reviewed in Ullrich, A. and Schlessinger, J., Cell 61: 203-211 (1990)).
Upon binding of these ligands, growth factor receptors undergo dimerization followed by the autophosphorylation of specific tyrosine residues.
Among the intracellular milieu of proteins are molecules with defined biological functions that serve as substrates for ligand-activated tyrosine kinase receptors.
The experimental evidences accumulated in the last few years indicate that, upon binding to the receptor, these substrate molecules switch into an activated form and become part of the critical signaling pathway used by growth factors to control cell proliferation.
A number of the cytoplasmic molecules that mediate cellular response to growth factors have been shown to interact with activated receptors through their SRC homology region 2 (SH2) domain (Koch, C. A., et al., Science 252: 668-674 (1991)).
The SH2 domain is a conserved protein module of approximately 100 aminoacids which is found in a remarkably diverse group of cytoplasmic signaling proteins.
Proteins with SH2 domains frequently possess another distinct sequence of about 50 residues, the SH3 domain, which is also implicated in the regulation of protein-protein interactions during signal transduction (see Clark, S.G., et al., Nature 356: 340-344 (1992) and references therein).
Receptor autophosphorylation following ligand binding acts as a molecular switch to create binding sites for the SH2 domain of the cytoplasmic signaling proteins (Anderson, D., et al., Science 250: 979-982 (1990)), which thereby become targets for activation.
SH2 domains directly recognize phosphotyrosine (Matsuda, M., et al., Science 248: 1537-1539 (1990)).
However, high affinity binding of an SH2 domain requires that the phosphotyrosine be embedded within a specific amino acid sequence, as originally suggested by an examination of SH2-binding sites (Cantley, L. C., et al., Cell 64: 281-302 (1991)).
For example, the SH2-containing proteins phosphatidylinositol (PI) 3-kinase, the Ras GTP-ase-activating protein (Ras GAP) and phospholipase C-.gamma. (PLC-.gamma.) each bind to different autophosphorylation sites of the .beta. receptor for platelet-derived growth factor (Kashishian, A., et al., EMBO J. 11: 1373-1382 (1992); Fantl, W. J., et al., Cell 69: 413-423 (1992)).
Autophosphorylation sites acting as specific docking sites for PI 3-kinase, PLC-.gamma. and Ras GAP, have been identified also in epidermal growth factor receptor (EGF-R), colony-stimulating factor 1 receptor (CSF-1R) and fibroblast growth factor receptor (FGF-R) (Cantley, L. C., et al., Cell 64: 281-302 (1991); Mohammadi, M., et al., Mol. Cell. Biol. 11: 5068-5078 (1991); Reedijk, M., et al., EMBO J. 41: 1365-1372 (1992); Rotin, D., et al., EMBO J. 11: 559-567 (1992)).
It has been demonstrated that relatively short peptide sequences corresponding to PDGF receptor phosphorylation sites inhibited the interaction between the activated PDGF receptor and PI 3-kinase (Escobedo, J. A., et al., Mol. Cell. Biol. 11: 1125-1132).
The residues immediately C-terminal to the phosphotyrosine, especially those at the +1, +2 and +3 positions, appear to provide selectivity for specific SH2 domains.
Thus, a recognition motif for the p85 submit of PI 3-kinase has been identified in the PDGF receptor: the sequence is Tyr-Met/Val-Xxx-Met (YMXM or YVXM) (SEQ. ID NOS: 1 and 2) wherein Xxx and X represent any amino acid residue in the three-letter or one-letter code respectively (Domchek, S. M., et al., Biochemistry 31: 9865-9870 (1992)).
The identification of new members of the SH2-containing molecules family as well as of the respective recognition motif is rapidly proceeding.
A direct involvement of some of these molecules in eliciting the biological response to a ligand has been demonstrated. This is the case for the protein Grb2 which associates, through its SH2 domain, to both EGF receptor upon ligand stimulation.
Microinjection of Grb2 and H-ras protein into mammalian cells resulted in the stimulation of DNA synthesis and hence in a mitogenic effect (Lowenstein E. J., et al., Cell 70: 431-442 (1992)).
These results indicate that Grb2 plays a crucial role in the mechanism for growth factor control of ras signaling.
While most of the efforts in the last few years have been concentrated on the study of the interaction between cytoplasmic signaling proteins and the best characterized EGF and PDGF receptors, the authors of the present invention have focused their attention on the hepatocyte growth factor (HGF) receptor.
HGF, also known as Scatter Factor (SF), is a heterodimeric protein secreted by cells of mesodermal origin (Stoker, M., et al., Nature 327: 239-242 (1987); Weidner, K. M., et al., J. Cell Biol. 111: 2097-2108 (1990)).
The factor induces a spectrum of biological activities in epithelial cells, including mitogenesis, stimulation of cell motility and promotion of matrix invasion (Nakamura, T., et al., Biochem. Biophys. Res. Comm. 122: 1450-459 (1984); Stoker, M. et al., Nature 327: 239-242 (1987); Weidner, K. M., et al., J. Cell. Biol. 111: 2097-2108 (1990); Rubin, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. 88: 415-419 (1991)).
HGF/SF is also a morphogen in vitro (Stern C. D. et al., Development 110: 1271-1284 (1990); Montesano et al. Cell 66: 697-711 (1991)) and a potent angiogenic factor in vitro and in vivo (Bussolino, M. F., et al., J. Cell Biol. 119: 629-641 (1992)).
While the biological effect of HGF/SF varies depending on the target cell, the HGF/SF signal is mediated by a single receptor, the tyrosine kinase encoded by the MET proto-oncogene (for a review see Comoglio, P. M. in I. D. Goldberg and E. M. Rosen (eds), Hepatocyte Growth Factor--Scatter Factor (HGF/SF) and the C-Met Receptor, Birkhauser Verlag Basel/Switzerland).
The HGF receptor, also known as p190.sup.MET, is a heterodimeric receptor made of an extracellular .alpha. and a transmembrane .beta. subunit (Giordano, S., et al., Nature 339: 155-156 (1989)), both originating from proteolytic cleavage of a common single chain precursor of 170 kDa (Giordano, S., et al., oncogene 4: 1383-1388 (1989)).
NIH3T3 fibroblasts transfected with the human MET cDNA express functional receptors and respond to HGF/SF with increased motility and invasion of extracellular matrices (Giordano S. et al. PNAS, 90: 649-653 (1993)).
Uncontrolled tyrosine kinase activity of the HGF/SF receptor has been observed in transformed cell lines, following chromosomal rearrangements (Park et al., Cell 45: 895-904 (1986)), gene overexpression (Giordano S. et al., Nature 339: 155-186 (1989)), defective post-translational processing (Mondino et al., Mol. Cell. Biol. 11: 6084-6092 (1991)) and autocrine loop.
Overexpression of the receptor has been observed in a number of human tumors of epithelial origin (Di Renzo et al., Oncogene 6: 1997-2003 (1991); Di Renzo et al., Oncogene 7: 2549-2553 (1992); Prat et al., Int. J. Cancer 49: 323-328 (1991)).
This emphasizes the oncogenic potential of the HGF/SF receptor.
Little is known about the signal transduction pathways triggered by HGF/SF.
The pleiotropic biological response induced by the factor suggests that more than one mechanism may be activated. The present inventors have previously shown that the HGF/SF receptor associates in vitro, upon autophosphorylation, PI 3-kinase, ras-GAP, PLC-.gamma. and Src-related tyrosine kinases (Bardelli, A., et al., Oncogene 7: 1973-1978 (1992)).
Association of PI 3-kinase with the activated receptor has also been found in vivo, in cells stimulated by HGF/SF (Graziani, A., et al., J. Biol. Chem. 266: 22087-22090 (1991)).
Recently, the authors have also shown that HGF/SF activates Ras by increasing the turnover between its GDP- and GTP-bound state through the stimulation of a guanine nucleotide exchange factor (Graziani, A., et al., J. Biol. Chem. in press (1993)).
We have now found that the HGF/SF receptor associates with the proteins Shc and Gbr2. The mammalian gene Shc encodes three widely expressed overlapping proteins of 66, 46 and 52 kDa (p66.sup.Shc, p46.sup.Shc and p52.sup.Shc) containing a C-terminal SH2 domain and a N-terminal collagen-homology region (Pelicci et al, Cell 70: 93-104 (1992)). p46.sup.Shc and p52.sup.Shc are encoded by the same transcript by employing two different ATGs.
p66.sup.Shc is encoded by an alterantively spliced transcript (Migliaccio et al, in preparation). Experimental evidence indicates that Shc proteins are implicated in the transduction of signals generated by tyrosine kinase receptors. Among these, Shc proteins are rapidly tyrosine-phosphorilated in response to activation of the EGF (Pelicci et al, ibid) and PDGF (unpublished data) receptors, Erb-B-2 (Segatto et al., Oncogene 2105-2112 (1993)), Src and Fps (McGlade et al, Proc. Natl. Acad. Sci. U.S.A. 89: 8869-8873 (1992)). Overexpression of Shc proteins induces neurite outgrowth in PC12 pheochromocytoma cells, and this effect is blocked by the expression of a dominant-negative Ras mutant (Rozakis-Adcock et al., Nature 360: 689-692 (1992)). Upon cell stimulation by certain growth factors, Shc proteins form stable complexes with the Grb2/Sem5 adaptor (Lowenstein et al., Cell 70: 431-442 (1992); Rozakis-Adcock et al., ibid). The latter adaptor is known to activate Ras functions by recruiting SoS, a guanine nucleotide exchanger factor (Li et al., Nature 363: 85-87 (1993); Gale et al., Nature 363: 88-92 (1993); Rozakis-Adcock et al., Nature 363: 83-85 (1993); Egan et al., Nature 363: 45-51 (1993); Simon et al., Cell 73: 169-177 (1993); Oliver et al., Cell 73: 179-191 (1993)) to the membrane.
From the above it clearly appears that the interaction between an activated tyrosine kinase receptor and a cytosolic transducer molecule is a critical step in the signaling pathway leading to cell proliferation and motility.
Since these biological responses constitute the most peculiar characteristics of tumor growth and spreading, need for interfering in such interaction is recognized in the art.